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1.
Genet Med ; 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31578471

RESUMO

PURPOSE: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved. METHODS: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types ofSOX5 alterations. Functional consequences of selected substitutions were investigated. RESULTS: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated. CONCLUSIONS: This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

2.
Cancer Res ; 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31662325

RESUMO

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ε (IKKε) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKε substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKε activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNß. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKε in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. SIGNIFICANCE: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.

3.
Genet Med ; 21(9): 2036-2042, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30739909

RESUMO

PURPOSE: To define the clinical characteristics of patients with variants in TCF20, we describe 27 patients, 26 of whom were identified via exome sequencing. We compare detailed clinical data with 17 previously reported patients. METHODS: Patients were ascertained through molecular testing laboratories performing exome sequencing (and other testing) with orthogonal confirmation; collaborating referring clinicians provided detailed clinical information. RESULTS: The cohort of 27 patients all had novel variants, and ranged in age from 2 to 68 years. All had developmental delay/intellectual disability. Autism spectrum disorders/autistic features were reported in 69%, attention disorders or hyperactivity in 67%, craniofacial features (no recognizable facial gestalt) in 67%, structural brain anomalies in 24%, and seizures in 12%. Additional features affecting various organ systems were described in 93%. In a majority of patients, we did not observe previously reported findings of postnatal overgrowth or craniosynostosis, in comparison with earlier reports. CONCLUSION: We provide valuable data regarding the prognosis and clinical manifestations of patients with variants in TCF20.

4.
Cancer Cell ; 34(3): 439-452.e6, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30205046

RESUMO

Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição
5.
Amino Acids ; 49(8): 1325-1335, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28536844

RESUMO

Pancreatic cancer is one of the deadliest human malignancies and lack of effective diagnostic and therapeutic methods. Accumulating evidence suggests that the neurotensin (NT) and neurotensin receptors (NTRs) play key roles in pancreatic adenocarcinoma growth and survival. In this study, we not only evaluate the NTR1 expression in pancreatic cancer patient samples, but also explore the PET and fluorescence imaging of NTR1 expression in pancreatic cancer animal models. The NTR1 expression was evaluated by immunohistochemistry staining in clinical patient tissue samples with pancreatic ductal adenocarcinoma, insulinoma, and pancreatitis. The results showed 79.4% positive rate of NRT1 expression in pancreatic ductal adenocarcinoma, compared with 33.3 and 22.7% in insulinoma and pancreatitis samples, respectively. High NTR1 gene expression was also found in Panc-1 cells and confirmed by cell immunofluorescence. 64Cu-AmBaSar-NT and IRDye800-NT were synthesized as imaging probes and maintained the majority of NTR1-binding affinity. In vivo imaging demonstrated that 64Cu-AmBaSar-NT has prominent tumor uptake (3.76 ± 1.45 and 2.29 ± 0.10%ID/g at 1 and 4 h post-injection). NIR fluorescent imaging with IRDye800-NT demonstrated good tumor-to-background contrast (8.09 ± 0.38 × 108 and 6.67 ± 0.43 × 108 (p/s/cm2/sr)/(µW/cm2) at 30 and 60 min post-injection). Fluorescence guided surgery was also performed as a proof of principle experiment. In summary, our results indicated that NTR1 is a promising target for pancreatic ductal adenocarcinoma imaging and therapy. The imaging probes reported here may not only be considered for improved diagnosis of pancreatic ductal adenocarcinoma, but also has the potential to be fully integrated into patient screening and treatment monitoring of future NTR1 targeted therapies.


Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Insulinoma/patologia , Neoplasias Pancreáticas/patologia , Pancreatite/patologia , Receptores de Neurotensina/metabolismo , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Animais , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/metabolismo , Humanos , Indóis/metabolismo , Indóis/farmacocinética , Insulinoma/diagnóstico por imagem , Insulinoma/metabolismo , Masculino , Camundongos , Camundongos Nus , Neurotensina/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/metabolismo , Pancreatite/diagnóstico por imagem , Pancreatite/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Immunol Res ; 4(6): 520-30, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27068336

RESUMO

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520-30. ©2016 AACR.


Assuntos
Autofagia/fisiologia , Pancreatite/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Doença Aguda , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Proteína 5 Relacionada à Autofagia/genética , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/biossíntese , Benzamidas/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Ceruletídeo , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Ativação Enzimática/genética , Deleção de Genes , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia , Pancreatite/prevenção & controle , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas
7.
J Clin Invest ; 124(12): 5411-23, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25365225

RESUMO

Triple-negative breast cancers (TNBCs) are a heterogeneous set of cancers that are defined by the absence of hormone receptor expression and HER2 amplification. Here, we found that inducible IκB kinase-related (IKK-related) kinase IKBKE expression and JAK/STAT pathway activation compose a cytokine signaling network in the immune-activated subset of TNBC. We found that treatment of cultured IKBKE-driven breast cancer cells with CYT387, a potent inhibitor of TBK1/IKBKE and JAK signaling, impairs proliferation, while inhibition of JAK alone does not. CYT387 treatment inhibited activation of both NF-κB and STAT and disrupted expression of the protumorigenic cytokines CCL5 and IL-6 in these IKBKE-driven breast cancer cells. Moreover, in 3D culture models, the addition of CCL5 and IL-6 to the media not only promoted tumor spheroid dispersal but also stimulated proliferation and migration of endothelial cells. Interruption of cytokine signaling by CYT387 in vivo impaired the growth of an IKBKE-driven TNBC cell line and patient-derived xenografts (PDXs). A combination of CYT387 therapy with a MEK inhibitor was particularly effective, abrogating tumor growth and angiogenesis in an aggressive PDX model of TNBC. Together, these findings reveal that IKBKE-associated cytokine signaling promotes tumorigenicity of immune-driven TNBC and identify a potential therapeutic strategy using clinically available compounds.


Assuntos
Neoplasias da Mama/metabolismo , Quimiocina CCL5/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-6/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Animais , Benzamidas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quimiocina CCL5/genética , Feminino , Humanos , Quinase I-kappa B/genética , Interleucina-6/genética , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Pharmacogenomics ; 15(11): 1507-18, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25303301

RESUMO

Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma de Pulmão , Animais , Autofagia/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Citocinas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases raf/metabolismo
9.
Cancer Res ; 74(7): 2082-93, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24525741

RESUMO

Aberrations of Notch signaling have been implicated in a variety of human cancers. Oncogenic mutations in NOTCH1 are common in human T-cell leukemia and lymphomas. However, loss-of-function somatic mutations in NOTCH1 arising in solid tumors imply a tumor suppressor function, which highlights the need to understand Notch signaling more completely. Here, we describe the small GTPase RhoE/Rnd3 as a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) that arise in skin epithelia. RhoE is a transcriptional target of activated Notch1, which is attenuated broadly in SCC cells. RhoE depletion suppresses Notch1-mediated signaling in vitro, rendering primary keratinocytes resistant to Notch1-mediated differentiation and thereby favoring a proliferative cell fate. Mechanistic investigations indicated that RhoE controls a key step in Notch1 signaling by mediating nuclear translocation of the activated portion of Notch1 (N1IC) through interaction with importins. Our results define RhoE as a Notch1 target that is essential for recruitment of N1IC to the promoters of Notch1 target genes, establishing a regulatory feedback loop in Notch1 signaling. This molecular circuitry may inform distinct cell fate decisions to Notch1 in epithelial tissues, where carcinomas such as SCC arise.


Assuntos
Carcinoma de Células Escamosas/patologia , Receptor Notch1/fisiologia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Carcinoma de Células Escamosas/química , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Queratinócitos/metabolismo , Camundongos , Receptor Notch1/análise , Neoplasias Cutâneas/patologia , Proteínas rho de Ligação ao GTP/análise , Proteínas rho de Ligação ao GTP/genética
10.
Cancer Discov ; 4(4): 452-65, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24444711

RESUMO

Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)-related kinases Tank-binding kinase-1 (TBK1) and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.


Assuntos
Comunicação Autócrina , Benzamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Quimiocina CCL5/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Camundongos , Neoplasias Experimentais , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo
11.
Cell Rep ; 3(3): 747-58, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23453972

RESUMO

Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKß, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Serina-Treonina Quinases/química , Ubiquitinação , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo
12.
Am J Pathol ; 177(3): 1176-86, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20651226

RESUMO

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down-regulation and/or mutations of integrin alpha7 (ITGA7) in prostate cancer, liver cancer, soft tissue leiomyosarcoma, and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two-hybrid analysis revealed that the C-terminus of ITGA7 interacts with high temperature requirement A2 (HtrA2), a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, whereas down-regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site-directed protease-null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears to protect against cell death, since depleting laminin beta2 with a small-interfering RNA significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.


Assuntos
Antígenos CD/metabolismo , Apoptose/fisiologia , Cadeias alfa de Integrinas/metabolismo , Proteínas Mitocondriais/metabolismo , Próstata/metabolismo , Serina Endopeptidases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Laminina/metabolismo , Masculino , Técnicas do Sistema de Duplo-Híbrido
13.
Am J Pathol ; 173(6): 1758-67, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18988800

RESUMO

MCM7 is a critical component of the DNA replication licensing complex that controls DNA replication in both yeast and Xenopus. Our previous studies have indicated that MCM7 is both amplified and overexpressed in metastatic prostate cancer. In this study, we found that MCM7 interacts with the androgen receptor (AR) with high affinity both in vitro and in vivo. We identified the AR-binding motif for MCM7, comprised of amino acids 221 to 248, and the MCM7-binding motif for the AR, comprised of amino acids 426 to 475. AR stimulation with high doses of the synthetic androgen R1881 led to a decrease in MCM7 binding to genomic DNA, a reduction of DNA synthesis, decreases in the number of cells progressing through S phase and cell proliferation, whereas low doses produced an increase in the DNA licensing activity of MCM7 and higher levels of cell proliferation. In addition, the MCM7/AR interaction down-regulated MCM7 expression. The gene transcription or repressor activity of AR is dependent on its interaction with MCM7 because either a mutant AR defective in its interaction with MCM7 or a MCM7 knockdown primarily eliminated AR effects on gene expression. Thus, this study reveals a novel mechanism by which AR and MCM7 facilitate each other's function, suggesting that AR-independent activation of MCM7 may be a mechanism by which prostate cancers bypass therapeutically induced AR blockade.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Metribolona/metabolismo , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Proteínas Recombinantes de Fusão/genética , Xenopus laevis
14.
Brain Res ; 1114(1): 1-10, 2006 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-16930567

RESUMO

Prolonged or excessive exposure to corticosterone leads to neuronal damages in the brain regions, including hippocampus. We reported that astrocyte-conditioned medium (ACM) protected the neurons of the primary hippocampal cultures against the corticosterone-induced damages. Corticosterone added to the cultures resulted in a significant number of TUNEL-positive cells. However, corticosterone-induced TUNEL labeling was suppressed as for ACM-cultured neurons. To delineate the molecular basis underlying the neuroprotection of ACM, we assessed the activation of ERK1/2 and (PI3-K)/Akt signal pathways in response to corticosterone-induced neuronal damages. Western blot test revealed that corticosterone increased the phosphorylation of ERK1/2 and PI3-K/Akt in hippocampal neurons grown in Neurobasal medium supplemented with B27 and 500 microm L-glutamine (NBM+). Interestingly, the increase of phospho-ERK1/2 and Akt levels was much pronounced and the time course of phosphorylation was altered in ACM, suggesting that both signaling pathways might participate in ACM protection. Furthermore, the selective inhibitor of Akt, rather than ERK1/2, blocked the neuroprotective activity against corticosterone in ACM-cultured neurons. In summary, our data showed that ACM had a potent neuroprotective effect in cultured neurons. PI3-K/Akt signal pathway, but not ERK1/2, was involved in the protective activity against the corticosterone-induced damages.


Assuntos
Astrócitos/química , Corticosterona/efeitos adversos , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Interações de Medicamentos , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
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